Retroviruses are the causative agents for an increasing number of diseases of higher organisms including: AIDS, HIV, various leukemias, feline leukemia, murine leukemia, several avian leukemias, various sarcomas of mice, rats, monkeys, birds, and cats, and other lymphotrophic diseases of man, including Adult T-Cell leukemia. Acquire Immune Deficiency Syndrome (AIDS), the recently most noteworthy of these diseases, is caused by a retrovirus which has been called HTLV-III, LAV, RAV or most recently HIV. Coffin et al, Science, 232:697 (1986). HIV is one of a group of retroviral diiseases which attacks the T4 lymphocytes thereby destroying the body's immune system. Anderson, Science, 226:401-409 (1984); Weiss, In RNA Tumor Viruses-II, vol. 2, Cold Spring Harbor Laboratory, pp. 405-485 (1985). The disease is uniformly fatal and no cure has been developed which either kills the virus in situ or replaces the lost elements of the body's immune system. Some experimental drugs such as HPA-23, azidothymidine and suramin show limited effects in stopping the virus, and immunimodulators such as thymostimulin and isoprinosine hopefully will bolster the patient's malfunctioning immune system, but to date there is no proven therapy or cure for the AIDS patient. It is also unlikely that a traditional vaccine for the virus will be developed for quite some time due to the wide variation in antigenicity of various strains of the virus.
Retroviral diseases differ from many other viral diseases in that the infective agent, a retrovirus, eventually becomes integrated in the host cell's genome. The retrovirus inserts its genome into a host chromosome, such that its genetic material becomes part of the genetic makeup of the infected cell, and is then replicated with the cell as the cell divides and multiplies. It is this characteristic which makes retroviruses especially persistent and immune to traditional anti-viral treatment. There is as yet no way to kill the retrovirus without killing the host cell. Thus, there is no proven cure, nor is there any proven effective vaccine or pharmacological agent against any retroviral disease.
Details of the life cycle and replication of retroviruses are discussed at length in Weiss et al, RNA Tumor Viruses, vols. 1 and 2 (Cold Springs Harbor Laboratory 1984), which is incorporated herein by reference in its entirety. FIG. 1(B) summarizes a model of a retrovirus life cycle. The life cycle of retroviruses is unique among viruses. The cycle begins when an infectious particle enters a host cell and releases two identical RNA molecules. These molecules are "reverse transcribed" by special viral enzymes to produce double-stranded DNA which circularizes and inserts into the host chromosome. FIG. 1(A) summarizes a model of the synthesis of double-strand DNA from viral RNA. The inserted DNA virus or "pro-virus" is structurally very similar to a normal host gene. It is transcribed to produce RNA, like any host gene. This RNA can then be processed in three ways: a) it can be directly translated into certain viral proteins, b) it can be processed and spliced, and then translated to produce other viral proteins, or c) it can be packaged, along with various viral proteins to make a newly infectious particle. In the case of HIV, the infectious particles continuously "bud off" the infected cells and bind to uninfected cells, beginning the cycle over again.
The retroviral particle which is the infectious agent contains in its interior two single-stranded positive-sense viral RNA molecules each between 7,000 to 11,000 nucleotide bases in length. These viral RNA's combine with certain viral proteins to form a viral core; the core being surrounded by a membrane. Imbedded in the membrane are viral glycoproteins which can specifically bind the viral particles to the appropriate host cell system. The viral core is assembled within the host cell and exits from the host cell, taking some of the host's membrane with it. Hence the membrane of the viral particle is derived directly from the host cell. The particle travels to an uninfected host cell, and due to the glycoprotein on its exterior binds to the new host cell and the life cycle repeats. Once the virus enters the cell, it is disassembled, releasing the two identical viral RNA molecules. These molecules are each composed of a sequence having specific functional regions making up the viruses "genomic structure".
The genome of any retrovirus is divided into three regions: the 5' terminus, the 3' terminus and a central region containing genes coding for proteins. The 5' terminus is further divided into four functional regions: the terminal redundancy (R), a unique sequence (U5), the primer binding site (PB- or PBS) and an untranslated sequence (L). The L region may contain a splice donor site for subgenomic mRNA. The 3' terminus is further divided into three functional regions: the primer-binding site for positive strand DNA synthesis (PB+ or PBS), a unique sequence (U3) and another copy of the terminal redundancy (R). The U5, U3 and R regions are sometimes collectively referred to as the Long Terminal Repeat (LTR) region. Components of the LTR region are involved in integration of the retroviral genome into the genome of its host. All retroviruses contain these highly conserved regions. These regions are further described by Weiss et al, supra, pp. 262-296.
The production of DNA from the infectious RNA occurs by a complex process called reverse transcription. The viral reverse transcriptase enzyme first complexes with a specific tRNA molecule supplied by the host cell. For example, in the case of the AIDS-related virus, it is lysine tRNA which complexes with the reverse transcriptase. The 3' end of the tRNA molecule remains free to hybridize with the primer binding site (PBS) of the retroviral genome. This is a sequence within the virus, which is complementary to the 5' end of the tRNA. Once the virus/enzyme/tRNA complex has been formed, the enzyme can make a new DNA molecule, using the RNA virus as a template, and using the tRNA as a "primer". As the process proceeds, the RNA of the resulting RNA/DNA complex is degraded, leaving single-stranded DNA. This process begins internally at the PBS site and proceeds to the 5' end of the RNA virus, where the process is stalled and regresses slightly, leaving a single-strand DNA "sticky end". At this point the enzyme/DNA complex has to "jump" to a new template at the 3 ' end of the virus. This jump, termed the first jump, is possible because the newly synthesized DNA is complementary to the other R region at the 3' end of the virus. After this jump, reverse transcription continues around to the point of the primer binding site.
After the "first jump" and while reverse transcription continues, second-strand DNA synthesis begins from the poly-purine site upstream of the U3 region. This DNA second-strand synthesis continues in the opposite direction from the first-strand DNA synthesis and proceeds through the primer binding site. The RNA primer molecule is consequently degraded, leaving a short residual region of second-strand DNA extending from the region of double-strand DNA. At this point the enzyme/DNA complex needs to make a "second jump" to a new template, this time jumping to the "sticky end" of the second strand DNA. This is possible because of complementation between the first and second strand DNA molecules in the region of the primer binding site. After hybridization of the complementary ends, reverse transcription can continue using the second-strand DNA as a template. This subsequently results in displacement of the first strand DNA, past the site of the first jump, up to the point where the second strand synthesis begins. Second-strand synthesis which was stalled at the PBS site prior to the second jump, can also continue after this jump, and proceeds to the 5' end of the first-strand DNA. The result of this process is a double-stranded DNA molecule with additional redundancies at both ends. Note that the DNA genomic structure differs from the RNA genomic structure in having a redundant U3 region added to the 5' end, and a redundant U5 region added to the 3' end. This occurs because the reverse translation process copies more than one full length of the RNA genome. Note also that this genomic structure now resembles a normal gene, with U3 being the promoter, with structural genes in the center, and a U5 tail.
The exact process of how the DNA virus inserts into host chromosomes is not known. It is known that the DNA virus first becomes a circle, and that this involves the short inverted repeat sequences at the ends of the virus. These inverted repeats may be involved in some form of DNA hybridization which brings the ends of the virus together, allowing circularization. Subsequently, insertion into the chromosome is generally assumed to be mediated by an enzyme which recognizes the splice site in the circle and directs insertion of a single copy of the virus into a random site within the host chromosome.
The transcription of viral DNA from the DNA pro-virus within a chromosome occurs in a manner similar to the transcription of any host gene. The U3 region functions as a polymerase II promoter and transcription begins at the beginning of the R region. The U3 promoter like eukaryotic promoters generally requires a transcriptional activator protein, which turns the promoter "on". Transcription proceeds through most of the pro-virus and is terminated at the end of the 3' R region. As a result, the transcript is a recreation of the smaller and infectious single-strand RNA genome. A poly-A tail is attached to the 3' end of this RNA and the 5' end is capped, making this molecule similar to normal host messenger RNA.
The RNA which is transcribed from DNA can be directly translated into protein, like any mRNA within the host. The GAG and Pol proteins are produced in this way and are subsequently cleaved into several smaller proteins involved in viral assembly and reproduction. In such a case, the 5' end of the RNA binds to a ribosome and protein translation beings at the first AUG codon initiation triplet of the coding sequence closest to the 5' end of the RNA molecule. Translation is terminated by one of the standard "stop" codons. Genes which are distant from the 5' end of the viral RNA cannot be directly translated because of the intervening genes, such as GAG. Such intervening genes can be removed by a splicing process which involves breaks at specific sites in the RNA molecule, and re-ligation of the appropriate pieces. In this case, the 5' end of the RNA molecule is unchanged, and binds to the ribosome as before, but now the first AUG codon where translation begins is not at the beginning of the GAG sequence, but at the beginning of some other coding sequence further downstream.
Some viral RNA is not translated into protein but rather is packaged into infectious viral particles. Such packaging involves the binding of certain viral proteins to specific sequences of the viral genome. For example, in the RSV viral system, it is part of the GAG sequence which is one of the parts of the genome which binds to and is recognized by such proteins and have been shown to be necessary for packaging of the RNA. The RNA which is packaged into viral particles does not appear to be reverse-transcription-competent until "maturation" of the particle, i.e., after it has existed away from the host cell.
All retroviruses, including HIV, once inserted into the host chromosome, must have their genes translated into viral proteins. If viral proteins are not abundant, the retrovirus cannot efficiently propagate to other cells and is not cytopathic to the infected host cell. Dayton et al, Cell, 44:941-947 (1986); Fisher al, Nature, 320: 367-371 (1986). Such proteins are not produced without the proper functioning of certain viral regulatory proteins. One of the key DNA/RNA-binding regulatory proteins for the retrovirus HIV is the TAT protein. Keegan et al, Science 231: 699-704 (1986). The TAT protein is essential to protein translation of HIV, and possibly also involved in RNA transcription. It is apparent that the TAT protein recognizes and binds to the nucleic acid sequence corresponding to the 5' end of the R region. A second activator gene ART has also been shown to be important in HIV translation. Sodroski et al, Nature, 321: 412-417 (1986). DNA/RNA binding of the previously described activator proteins is essential to HIV replication. Therefore, introducing genes into host cells, i.e., gene therapy for humans or germline transformation for animals, which will code for modified proteins of the retrovirus which compete or interfere with TAT or ART, will effectively block retrovirus replication.
Past research efforts have been predominantly confined to two traditional anti-retroviral approaches: immunological prevention and pharmacological therapy. Unfortunately, neither of these approaches appears to be very promising for control of retrovirus diseases. At best, an effective vaccination might reduce risk of infection in healthy individuals, but it would not be expected to cure an infected individual. Also, chemical repression of virus diseases has not generally been effective in eradicating any persistent virus, and certainly would not be expected to eradicate a retrovirus. Anti-viral chemicals tend to slow the progress of a virus and to bolster native defense mechanisms, but chemical treatments must be continuously applied and typically have undesirable side effects.
For these reasons, it is doubtful that any retroviral disease can be cured by the traditional anti-viral approaches. An alternative approach to inhibiting retrovirus replication is genetic inhibition by introducing nucleic acid constructs into host cells, i.e., gene therapy or germline transformation, which will confer cellular resistance by hybridization interference.
The inhibition or modulation of the various steps in the retroviral replication process by DNA or RNA which will hybridize and block viral sequences has been termed "hybridization interference". Green et al, ANN. REV. Biochem., 55:569-97 (1987), which is incorporated herein by reference. There are essential steps in retrovirus replication which require nucleic acid hybridization, Gilboa et al, Cell, 6: 93-100 (1979). If any of these replication steps are blocked by pre-binding of the essential sites in the retrovirus genome; or binding of proteins or other cellular constituents in the retrovirus genome, to molecules coded for by genetically engineered nucleic acid sequences in the host cell the retrovirus replication process can not proceed. Note, that "Hybridization Interference" has also been referred to as an "Anti-sense approach". Green et al, ANN. REV. Biochem., 55:569-97 (1987). However, an ambiguity exists in that "sense" and "anti-sense" only apply to sequences coding for proteins, and nucleic acid constructs are disclosed herein which target retrovirus sequences not coding for proteins. Consequently, as used throughout the specification and appended claims, "Hybridization Interference" or "Anti-sense RNA" should refer to the use of RNA or DNA to bind with nucleic acid, protein or other cellular constituents to inhibit retrovirus replication.
The effectiveness of the anti-sense RNA approach has been demonstrated in several model viral systems. It was demonstrated in the SP bacteriophage system that certain messenger-RNA-interfering complementary RNA (micRNA) can have very significant anti-viral effects, as seen by reduced plaque number and plaque size, Coleman et al, Nature, 315: 601-603 (1985).
In addition, it has been suggested that the replication and cell transformation of the Rous Sarcoma Virus (RSV) was inhibited by a specific synthetic tridecamer oligodeoxynucleotide, Zamecnik and Stephenson, Proc. Natl. Acad. Sci., 75: pp. 280-2BB (1978). The synthetic complementary tridecamer was introduced extracellularly into the cytoplasm of chick embryo fibroblast cells infected with RSV virus, thereby blocking RSV replication by hybridization competition. However the tridecamer was not incorporated into the host genome or any other genetic vehicle, such that neither the sequence, nor an equivalent coding sequence, would replicate in the cell. This is a chemotherapeutic approach to inhibiting virus replication, and not gene therapy.
Another publication has shown that synthetic exogenous oligodeoxynucleotides complementary to regions of the HIV genome inhibit virus replication and gene expression in cultured cells. Sequences of exogenous synthetic oligodeoxynucleotides 12, 20, and 26 nucleotides in length were tested on infected cells, Zamecnik et al, Proc. Natl. Acad. Sci., 83:4143-4146 (1986). Again, the oligodeoxynucleotides are exogenous and were not incorporated into the host genome or another vehicle which would provide for the replication or maintenance of the tridecamer.
Finally, the anti-sense RNA-mediated inhibition on the replication of avian retrovirus in cultured cells was suggested using natural gene sequences derived from the neomycin resistant gene of the bacterial transposable element Tn5, To et al, Molecular and Cellular Biology, vol. 6, No. 12, pp. 4758-4762 (1986).
In the field of human medicine, altering the genotype of the host has not been a desirable method of fighting infectious disease. However, it is now believed that gene therapy will be possible in the relative future, Anderson, Science, 226:401-409 (1984). As a result, application of the anti-sense RNA approach within the field of medicine may be possible. Presently available gene therapy techniques are only effective for the genetic modification of bone marrow and blood cells. Because of this limitation, the projected use of gene therapy has generally been assumed limited to the correction of rare hereditary gene defects where such defects center in bone marrow or blood cells. Despite these limitations there are certain pathogens of the blood for which conventional defenses appear inadequate, and where the use of anti-sense RNA inhibition might be feasible. Many of the cells that are infected by retroviruses are derived from hematopoetic stem cells. If these stem cells can be altered by the incorporation of genes or other nucleic acid sequences which will synthesize RNA molecules that are antagonistic to virus propagation, an efficient method to both effectively prevent and to treat these retroviral diseases will be apparent. Further, if the expression of the RNA inhibiting genes can be regulated in the desired cells, it has application to other genetic diseases.
It would therefore be desirable to provide methods and compositions for producing RNA which is complementary or corresponding to an essential retroviral hybridization site within the retrovirus genome selected from the group consisting of the LTR region, the U5 region, the U3 region, the R region, the PBS region, the AUG start codon regions, the polyP region, RNA splice sites, the leader region, the TAT splice site, the ART splice site and the cap site which would be effective to inhibit retroviral replication.
Another objective is to provide methods and compositions for expression in a host cell system of a synthetic double-strand DNA fragment coding for an RNA fragment complementary or corresponding to an essential retroviral hybridization site within the retrovirus genome selected from the group consisting of the LTR region, the U5 region the U3 region, the R region, the PBS region, the AUG start codon regions, the polyP region, RNA splice sites, the leader region, the TAT splice site, the ART splice site and the cap site, without adverse side effects to the host cell resulting from such gene expression.